ABSTRACT We propose to address several important questions relevant to achieving a functional cure of HIV infection in individuals on suppressive antiretroviral therapy (ART). Specifically, we will examine the timing and mechanisms that establish the active or infectious reservoirs in primary human immunodeficiency virus type 1 (HIV-1) infection; identify the mechanisms by which active HIV reservoirs persist for many years under ART, identify the cells that harbor active reservoirs, and; discern whether reservoirs in individuals initiating ART during primary infection persist in antigen-specific cells and are thus amenable to activation with their target antigens for destruction strategies. Our subject population derives from a unique and long standing cohort of individuals enrolled while in the first few days to months of HIV infection, who went onto suppressive ART prior to 4 months from the date of infection and have had intensive specimen collection during follow-up for up to ~14 years. One hundred and one subjects who initiated ART in Fiebig stages I-V, and remained on ART and virologically suppressed for at least 2 years, form the primary set of subjects for study. Specimens from 4 additional subjects who started ART in chronic HIV infection and experienced a viral rebound when ART was interrupted will be also be used to address one of our questions. We will quantify virus in plasma RNA, as total viral DNA and as 2LTR circular unintegrated viral DNA in peripheral blood mononuclear cells (PBMC), and examine the infectious capacity of proviral DNA in resting CD4+T cells. We will also determine the contribution of proliferating HIV-infected cells, and the relationship between disruption of cellular genes regulating the cell cycle by HIV integration into the chromosome, to the persistence of active reservoirs in blood and in specific subpopulations in PBMC, bronchioalveolar lavage fluids and biopsies of gut-associated lymphoid tissues. Additionally, we will determine whether the putative reservoirs we identify allow HIV to remain active by virtue of inadequate intracellular levels of antiretroviral drugs. Our specific aims are to determine: 1) The mechanisms and kinetics of establishment of active HIV reservoirs; 2) the relative contributions of (A) proliferating HIV- infected cells, and (B) low-level viral replication to the persistence of active reservoirs during ART, and; 3) the proportion of HIV reservoir cells composed of virus-specific CD4+T cells.